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    公开号:SE534150C2
    申请号:SE0850111
    申请日:2007-05-02
    公开日:2011-05-10
    发明作者:Jon Odorico；Xiaofang Xu
    申请人:Wisconsin Alumni Res Found；
    IPC主号:
    专利说明:

    [5] Embryonic stem cells (singular: ESC, plural: ESCs) from primates and humans have been isolated and proliferated in culture. Embryonic stem cells are stem cells that can be maintained without restriction by self-renewal and proliferation in culture, but which can also maintain the ability to differentiate spontaneously into cells of different developmental lines. In non-selective conditions, it has previously been shown that a wide variety of stem cells, including mouse and human ESC, will differentiate spontaneously into cells of your developmental lines, including the pancreatic developmental line. It has been previously shown that such differentiated cells can express the pancreatic duodenal homeobox (PDX1) gene, which is a transcription factor that determines the pancreatic developmental line and can also express the insulin hormone.
    [6] Culture systems that enable the spontaneous differentiation of hESC into insulin-staining cells have been reported (Assady, S. et al., Insulin production by human embryonic stem cells. Diabetes 50, 1691-1697 (2001); and Segev, H. et al, Differentiation of human embryonic stem cells into insulin-producing clusters. Stem Cells 22, 265-274 (2004)). However, these studies have neither studied endodoric marker expression nor shown the development of cells that have stereotypic properties of ß-cells: simultaneous expression of C-peptide and pancreatic duodenal homeobox (PDX1), which is required for pancreatic formation and co-activates the insulin promoter (Jonsson, J. et al., Insulin promoter factor 1 is required for pancreatic development in mice. Nature 371, 606-609 (1994)). Because non-ß cells such as neuronal cells can express insulin (Sipione, S. et al., Insulin expressing cells from differentiated embryonic stem cells are not beta cells. Diabetología 47, 499-508 (2004)), and insulin present in the culture medium can taken up in other cell types in certain conditions in vitro (Raj agopal, J. e: al., Insulin staining of ES cell progeny from insulin uptake. Science 299, 363 (2003)), it is important that the endodernal and pancreatic origin of insulin-staining cells derived from hESC are ensured. 534 150
    [7] It was recently reported that spontaneous differentiation of human ESCs produced PDX1-1- / FOXA2 + cells and co-transplantation of these differentiated cells with mouse dorsal pancreas (E1,5) resulted in PDX1 + / insulin + cells, and co-staining of insulin and C-peptide. were observed (Brolen, GK et al., Signals from the embryonic mouse pancreas induce differentiation of human embryonic stem cells into insulin-producing beta-cell-like cells. Diabetes 54, 2867-2874 (2005)). This report showed that the cells of the pancreatic developmental line can be induced from spontaneously differentiating human ESCs using signals derived from embryonic pancreas. However, the experimental procedures would be impractical to adapt to a high throughput culture protocol and the nature of the molecular signals was not revealed in this study. In addition, unselected stem cell populations are tumorigenic, which means that they will induce non-malignant tumors known as teratomas in immunodeficient animals in the same way as undifferentiated ES cells do.
    [8] Several studies have evaluated the effects of growth factors on human ESC differentiation into endoderms (Schuldiner, M. et al., Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 97, 11307- 11312 (2000) and D'Amour, KA et al. Effective differentiation of human embryonic stem cells to the definitive endodenn. Nat. Biotechnol. 23, 1534-1541 (2005). Nevertheless, such a technical level that is reproducible cannot be high. efficient differentiation into pancreatic precursors and islet cells is routinely achieved.In addition, the insulin-producing cells generated by previously described methods are less responsive to glucose (ie less functionally mature) than adult human beta cells and are thought to have a phenotype more similar to immature beta cells .
    [9] Methods for differentiating human ESCs, or other human pluripotent cell types, into pancreatic or pancreatic islet cells have been discussed in the patent literature. However, as stem cell culture techniques progress, improvements in the techniques for culturing differentiated cell types are necessary for the final commercial use of differentiated cells. The prior art techniques reported for the cultivation of human ESC into pancreatic developmental line cells, while being suitable for laboratory scale studies, cannot be easily scaled up to reliably and consistently produce large numbers of pancreatic cell types for research or therapeutic use. Thus, a simple, reproducible culture method using defined components that promote islet cell differentiation from human pluripotent stem cells is a desirable contribution to the art.
    [10] The present invention can be broadly summarized as new methods for direct in vitro differentiation of human pluripotent stem cells into cells of the endodermal and pancreatic developmental lineages. The method comprises culturing the stem cells with an effective amount of a bone morphogenesis protein to induce differentiation in the direction of mesendoderm. These mesendoderm cells are further cultured to form embryoid bodies (singular: EB; plural EBs) which, under defined conditions, terminally differentiate into cells of the pancreatic developmental line.
    [12] A further feature of the direct differentiation methods described herein is the ability to isolate large-scale endodennial and pancreatic cells, such as beta cells.
    [13] In addition, the methods described herein can overcome one of the major obstacles to the possible use of stem cell-derived cells for transplantation, the tumor-forming nature of undifferentiated stem cells. These methods can be used to derive populations of pancreatic islet cells that do not form teratomas upon host transplantation.
    [14] In one aspect, isolated cell populations derived from human pluripotent stem cells that have been linearized to the mesendodermal and endodermal lines of development are described. The cells of this developmental line are characterized by their ability to further differentiate into pancreatic islet cells, preferably beta cells. In a related aspect, at least 50% of the tenninally differentiated cells of the pancreatic developmental line are characterized by their ability to simultaneously express at least one or fl era of insulin, C-peptide, and PDX1 markers.
    [15] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art of this invention. Although suitable methods and materials for practicing or testing the present invention are described below, other methods and materials similar or equivalent to that described herein may be used which are well known in the art.
    [16] Other objects and advantages of the present invention will become apparent from the following description.
    [17] FIG. 1 is a fate diagram summarizing the general procedure for producing pancreatic lineage cells from human ES cells.
    [18] FIG. 2, including Figures 2 (a-f), shows a time sequence of gene expressions: (a) secreted hCG protein levels from cultures of BMP4 / bFGF-treated hESC; (b) human chorionic gonadotropin (hCG), (c) brachyury (T), (d) oct4, (e) sox17 and (i) foxa2.
    [19] FIG. 3, comprising fi gures 3 (a-t) shows that BMP4-initiated mesendoderm differentiation characterized by expression of Brachyury, Foxa2 and Sox17.
    [20] FIG. 4, comprising fi gures 4 (a-g) shows the effects of BMP4 and bFGF treatment of hESC on endodene and pancreatic associated gene expression and on EB morphology. Figures 4 (a) RT-PCR and (b) Q-PCR analysis show changes in expression of endodene (sox17, foxa2, pdxI) and pancreatic-associated (pdxI, glut2, insulin) genes at EB14 for hESCs as either were untreated, treated with 50 ng / ml BMP4, or treated with both 50 ng / ml BMP4 and 300 ng / ml Noggin. Figure 4 (c) shows mRNA levels from either hESCs grown on MEF treated with 50 ng / ml BMP4 or hESCs grown on MatrigelTM treated with only 50 ng / ml BMP4, or treated with both 50 ng / ml BMP4 and 100 ng / ml bFGF and analyzed at the EB14 stage. Figures 4 (d-g) are representative phase contrast images of 14 days EB 534 150 suspension cultures from untreated or treated hESC as follows; (d) EBs from untreated hESCs grown on MEF; (e) EBs from BMP4-treated hESCs grown on MEF; (t) EBs from BMP4-treated BMP4-treated hESCs grown on MatrigelTM; (g) EBs from hESCs grown on MatrigelTM treated with both BMP4 and bFGF. Size indicator, 100 um. FIG. 5, comprising fi guras 5 (a-o), characterizes EBs or stage 2 cells. Figure 5 (ac) are diagrams showing expression levels of endodene and pancreatic-associated genes in hESC-derived EB 14. Figure 5 (do) shows immunostaining of EB14s where bFGF was added during the EB stage and more than 50% of the cells are stained with PDX1.
    [22] FIG. Figure 6 shows a time course of gene expression after inoculation of intact EB and culture for 14, 21 and 28 days in growth factor complementary serum-free ITSFNE media (stage 3). EBs were from untreated (-) or BMP4-treated (+) hESCs grown on MEF: The expression of genes marking early neural (soxl), mesendoderm (I), undifferentiated ESC (oct4), trophoblasts (human chorionic gonadotropin, hCG), defective endodern (foxa2, pdxI), liver (tyrosine aminotransferase, TA T), and cellular endocrine cell types (ngn3, insulin, glucagon, glut2) were analyzed by RT-PCR.
    [23] FIG. 7, including guras 7 (a-p), are microscopic photographs showing that PDX1 + / insulin + cells are present in cultures at stage 3, i.e. BMP4 treatment of undifferentiated cells, a 14 day EB formation period, and further differentiation of such EBs. Figures 7 (a-c) show that EBs sown in ITSFNE medium for 14 days show co-staining of PDX1 and insulin. Figure 7 (d-t) shows that most PDX1-positive cells no longer stain with KI67 at this stage. Figures 7 (g-i) show larger clusters of PDX1 + insulin + co-staining cells arising at EB 144-28. Figures 7 (j-1) show that no PDX1 or insulin staining is observed in cultures not previously treated with BMP4. Figures 7 (m-p) show that the cells co-stained with insulin, C-peptide and PDX1 at EB14 + 2 8. This pattern is indicative of normal beta cells. Size marker, 50 pm. 534 150 8
    [24] FIG. 8, including guras 8 (a-h), are photomicrographs showing that glucagon-positive and somatostatin-positive cells are present at EB 14 + 28 in cultures previously treated with BMP4. Figures 8 (a-d) show that glucagon-positive cells do not stain with C-peptide and PDX1, as would be expected of normal adult alpha cells. Figures 8 (e-h) show that somatostatin-positive cells do not co-stain with C-peptide but some appear to stain with PDX1 as would be expected of normal adult delta cells. Size marker, 50 um.
    [25] FIG. 9 is a graph showing how different concentrations of growth factors (BMP4 and bFGF) lead to different developmental lines: (left) high bFGF and low - BMP4 (or with Noggin to inhibit endogenous sources of BMP) maintains hESCs in undifferentiated status; (middle) high bFGF and medium BMP4 lead to differentiation into endodermal and pancreatic developmental laryngeal cells; and (right) low bFGF and high BMP4 lead to trophectoder / trophoblast cells.
    [26] FIG. 10, including Figures 10 (a-c), shows that MACS sorting for EpCAM + cells results in enrichment of EpCAM cultures and endoderm and pancreas-associated transcripts. (A) Differentiated hESC co-expresses EpCAM (green) and PDX1 (red). Almost all PDX1 + cells co-express EpCAM; some EpCAM + cells that do not sanitize PDX1 are also visible. (B) Time-to-change values from QPCR analysis of differentiated EpCAM-sorted cells compared to unsorted cells from two independent experiments. (C) FACS analysis of EpCAM expression after differentiation of hESC with the BNlP4 / bFGF protocol. The cells were analyzed before and after MACS sorting for EpCAM. The percentage of EpCAM + cells increases from ~ 35% to ~ 95% accompanying MACS sorting for EpCAM.
    [28] FIG. 12 is a table listing primer sequences used for quantitative and non-quantitative PCR analysis of gene expression. DETAILED DESCRIPTION OF THE INVENTION The present invention broadly relates to novel methods for the direct in vitro differentiation of pluripotent mammalian stem cells into cells of the pancreatic developmental line. The methods involve culturing the stem cells in the presence of an effective amount of bone morphogenesis protein to induce differentiation in the direction of mesendoderm. These mesendoderm cells are further cultured to form embryoid bodies (singular: EB, plural: EBs) enriched for definitive endoderm developmental lineage cells, which in defined conditions terminally differentiate: into cells of the pancreatic developmental lineage. Utilizing defined medium components that promote pancreatic cell differentiation, the methods described provide a simple, reproducible approach to enable large-scale production of pancreatic cell types for research and therapeutic purposes.
    [30] In an attempt to better understand the methods described herein and the surrounding scientific literature, it is pointed out that recent studies with human embryonic stem cells (singular: hESC; plural: hESCs) have begun to focus on differentiation of definitive endoderm as a first steps towards the development of cells of the pancreatic developmental line. Others have reported on the subject Activin A induction of definitive endoderm from hESC (see D'Amour, K. A., et al. (2005)). However, cells of the pancreatic developmental lineage were not induced by this protocol. In addition, preliminary results from tests with Activin A (at 5 ng / ml, 50 mg / ml or 100 ng / ml) in serum-free medium suggest that this treatment alone cannot induce pancreatic cell differentiation. This is not surprising given that Activin A has been shown to maintain pluripotency of hESCs in the absence of feeder cells (Beattie, GM et al., Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stein Cells 23, 489-495 (2005)). Other hESC studies evaluating pancreatic degeneration have antigenically been inconclusive with respect to the origin of insulin-staining cells or have required a period of in vivo growth under undefined conditions (Brolen. G.K. et al., (2005)). 534 150
    [31] With the aim of identifying culture conditions that promote efficient differentiation of ß cells from hESC, a series of pilot experiments were performed to test a number of growth factors, cytokines and developmentally relevant molecules, including FGF4 (10 or 50 ng / ml), retinoic acid (10 9 M), FGF 10 (10 ng / ml), activin A (5 ng / ml, 50 ng / ml or 100 ng / ml), cyclopamine (1 μM or 10 μM) and BMP4 (5 ng / ml, 50 ng / ml or 100 ng / ml) at different stages of hESC differentiation. Of the factors tested, BMP4 treatment of hESCs grown on MEF by a large margin gave the strongest potentiating effect on pdx1 expression.
    [32] The intercellular signaling molecule BMP4 is known to play an important role in the determination of fate and the creation of developmental lines in embryogenesis. Several studies in other mammals have shown that BMP4 inhibits early neurogenesis in ESC cultures and promotes pancreatic endoderm specification from non-destined endoderm (Kumar, et al., Signals from lateral plate mesoderm instruct endoderm towards a pancreatic fate.
    [33] Again, through experimentation, the findings have shown that BMP4 treatment of hESCs promotes mesendoderm differentiation and subsequently supports pancreatic differentiation. The authors' data, together with data from Xu et al., Were used to identify a dose effect of BMP4 on hESCs. It was found that treatment with 100 ng / ml alone transforms almost all the cells into trophoblast cells, while lower doses can direct differentiation of hESCs to the mesendoder and definitive endoderm.
    [34] It is known that certain in vivo inductive events mediated by rnesoderm-derived embryonic tissues (ie, notochord, dorsal aorta, lamina lateralis mesoderm) play an important role in the pattern formation of non-fatal anterior edema and specifying a pancreatic fate. , JM. & Melton, DA (2000); and Kumar, et al. (2003)). The inventors believe that effects from other cell types and seedlings may be necessary for further maturation of endoderm cells and production of PDX1 + cells from hESCs: The production of pancreatic cell types from ESCs is probably a process with steg your steps that probably involves the sequential steps of induction of de itiv nitive endoderm, pattern formation of endoderm and induction of pancreatic epithelium, each of which requires proper signals from the environment, such as soluble growth factors and cytokines, and possibly direct contact with other cell types. Others report that PDX1 + / FoxA2 + cells were produced when hESCs were in direct contact with MEF and insulin-producing cells were produced from PDX1 + 1FoxA2 + cells after in vivo exposure to mouse dorsal pancreatic signals (Brolen, et al., (2005)) . On the other hand, it was recently reported that highly purified mouse endodermal cells did not express pancreatic markers after short-term culture (Tada, S. et al., Characterization of mesendoderm: a diverging point of the degenerative endoderm and mesoderm in embryonic stem cell differentiation culture .
    [36] On the contrary, the researchers discovered that the use of a medium level of BMP4 results in the assumption of a mesendodermal fate (brachyury +) of many cells. The endodermal marker FoxA2 arises during the initial treatment of hESC with BMP4 and MEF or with BMP4 and bFGF. In addition, F oxA2 expression gradually increases throughout the differentiation process and is associated with channel-like structures in EB. E fi subsequent pclx] gene expression and the emergence of PDX1 + cells also occurred during the EB phase. Although FoxA2 expression is not unique to definitive endoderm, still (1) the proximity of the cells to brachyury + cells, (2) observed co-staining with Soxl7, (3) the absence of neural markers, such as soxl, (4) low amount of trophoblast markers in these EBs, and (5) concomitant expression of PDX1 so that the FoxA2 + cells are likely to represent pre-pancreatic endodema or endoderm-derived cell populations. These results suggest that using a procedure with fl your steps involving early BMP4 and bFGF induction of mesendodem with the fi nied culture conditions, illustrated in FIG. 1, the endnitive endodermal and pancreatic developmental line cells can be sequentially derived from hESCs. 534 150 12
    [37] Accordingly, in a broad embodiment of the invention, the method provides a method of culturing human pluripotent stem cells in a medium containing an effective amount of bone morphogenesis protein to induce differentiation to the mesendoderal direction. As used herein, pluripotent mammalian stem cells include primate and preferably human embryonic stem cells (hESCs) as described by Thomson et al. (Science 2821145, 1998).
    [39] As used herein, an effective amount of a BMP comprises a concentration of from about 10 ng / ml to about 100 ng / ml. It is pointed out that while higher doses of BMP4 have been shown to induce differentiation of human pluripotent stem cells towards trofectodernal developmental lines, it is shown herein that the cultivation of stem cell colonies with low to intermediate amounts of BMP4, preferably 50 ng / ml, works synergistically with MEF or high concentrations of bFGF, such as 100 ng / ml, thereby promoting differentiation of the stem cells in the mesendodermal direction.
    [40] As used herein, mesendodermal cells are defined by, and not limited to, expression of Brachyury and Oct4 nuclear transcription factor markers (see stage 1 in FIG. 1). Mesendodermal cells may also be characterized by expression of 534 '150 Wnt3 and FGF4 (D'Amour et al. 2005) and / or goosecoid (Gsc), E-cadherin, Mix1, and F oxA2, and possibly Sox17 (Yasunaga et al. Nat. Biotechnol. 2005).
    [41] In the second phase of this embodiment, the cultures are induced to form embryoid bodies from small pluripotent stem cell colonies in MEF-conditioned medium. The learning obligation is EBs intact and is surrounded by a layer of Visceral yolk sac (VYS) that expresses stage-specific embryonic antigen-IS (SSEA-3). The length of the growing time for this step varies from about 1 day to about 4 weeks, preferably 14 days. As used herein, EBs are three-dimensional structures of groups of cells that interact in such a way that they induce further differentiation among the cells of EB (see stage 2 in Figure 1). Suitable EBs include definitive endoderm cells with tubular structures, which include cells expressing Foxa2, Sox17 and PDX1. It is believed that these endodennial cells give rise to cells of the pancreatic developmental line. As used herein, the cells of the pancreatic developmental line include, for example, cells that co-express PDX1 and Nkx6.1, which are well known to represent either pancreatic epithelial progenitor cells or beta cells. These cells are the only two cell types in the body that express this combination of markers or PDX1, insulin, and C-peptide, which are well known to coexist in normal beta cells; or cells expressing somatostatin which are generally considered to represent delta cells.
    [42] Cells within EBs derived from BlV [P4-treated hESCs were characterized by immunostaining and quantitative PCR as containing a significant. subpopulation that co-expresses both PDX1 and Nkx6. The 1-markers (see Fig. 4).
    [43] In the third phase of this embodiment, the cells are seeded from EBs into tissue culture plates in a serum-free medium (without fetal calf serum (FB S)) intended to induce terminal differentiation into cells of the endocrine developmental line. In this step, the length of time the cells are grown varies from about 7 days to about 56 days, preferably 28 days. Suitable terminally differentiated cells are characterized by the simultaneous expression of insulin, C-peptide and PDX1. Other cell types of the endocrine developmental line, such as glucagon-expressing cells and somatostatin-expressing cells, also arise in this context and in these regions of the cultures. A significant portion of the PDX1 + cells at these stages (stage 2 and stage 3) can be seen to co-express the cell surface marker epithelial cell adhesion molecule (EpCAM).
    [44] As used herein, serum-free medium means serum-free DMEM / F 12 (17.5 mM glucose) medium with ITS supplement (BD, 5 μg / ml insulin + 5 μg / ml transferrin + 5 ng / ml selenic acid), ng / ml FGF7 (R&D), 10 mM nicotinamide (Sigma), 10 nM exendin-4 (Sigma), and 2 g / L BSA (Sigma). Because of this, the serum-free medium is called ITSF NE for insulin-transferrin-selenium-FGF7-nicotinemide-exendin 4 (see stage 3 in fi g. 1). A suitable concentration for exendin-4 ranges from about 0.1 mM to about 1 mM. In addition, the concentration of nicotinamide can vary between about 1 and about mM.
    [45] Alternatively, in a related and more suitable embodiment, the human pluripotent stem cells may be grown initially without feeder cells, conveniently on MatrigelTM. When hESC was grown on Matrigel, the authors discovered that in addition to inducers of the bone morphogenetic signaling pathway, an effective amount of bl broblast growth factors (FGF) was needed to induce the cells to differentiate in the mesendodermal direction. The FGF concentration varies from about 10 to about 200 ng / ml. A suitable bFGF concentration is 100 ng / ml. At this stage, the inventors also discovered that the MEFs and factors they produce or produce can replace the function of FGF2 in this induction protocol. Adding bFGF to the cultures at stage 2 leads to essentially celler er cells of the endodennial and pancreatic developmental lines, which express the appropriate markers. The medium can be supplemented with other suitable fi bridge blast growth factors, such as FGF2. To characterize the cells of these three developmental stages, an RNA expression time course for a selection of endoderm and pancreatic-associated genes was performed and described below.
    [46] In some embodiments, the invention is also directed to methods of deriving cell populations enriched in pancreatic cells, preferably beta cells that do not 534 '150 form teratomas upon transplantation into hosts. Accordingly, it is contemplated that stage 3 cells of pancreatic developmental lineage may be made non-tumorigenic by sorting cells on the basis of cell surface markers as described in published U.S. application no. 2005 0260749 by the inventors. Specifically, the differentiated cells are sorted on the basis of positive expression of the epithelial cell adhesion molecule (EpCAM), a cell surface marker whose expression can be used for positive selection to identify human cells that are destined for the endodermal, pancreatic or forward developmental lines. To perform this selection, any instrument capable of sorting individual cells such as a fluorescence-activated cell sorter (FACS) or magnetically activated cell sorter (MACS) should be able to be adapted for use in this variety of cell sorting procedure. Using EpCAM / MACS selection protocols to remove undifferentiated cells left in later stages of culture, but also by using antibodies to cell surface markers of undifferentiated stem cells such as SSEA3 or SSEA4, the researchers have discovered that the tumorigenic tendency of stem cell cultures can be reduced.
    [47] It is clear from the stem cell literature that upon injection into immunocompromised mice, undifferentiated ES cells will form teratomas, which are non-malignant growths or tumors consisting of many different tissue types in a poorly organized structure. While the creation of teratomas is not considered life-threatening for the host, the teratomas can grow to large size, be intimidating and wasteful of metabolic energy for the host. A characterization of teratomas formed by human ES cells is found in Gertow et al., Stem Cells and Development, 132421-435 (2004). Finally, if human ES cells are to be used for cell or tissue transplantation, it is probably preferred that the cells thus delivered to the body be free of tumor-forming ability. In the art, the main techniques that have been described are to eliminate this ability based on inserting exogenous gene constructs into the ES cells and then selecting for differentiated cells based on expression characteristics of the introduced genes. However, the use of exogenous genes introduced into human ES cell cultures entails a different set of safety problems that should preferably be avoided. 534 150 16
    [48] By combining cell surface sorting technology with the direct differentiation procedures described herein, the inventors expect to be able to produce, on a practical level, stem cell-derived cell cultures that are not tumor-forming and that do not form teratomas. Despite the risk of recurrence, and to avoid misunderstandings, the use of the phrase tumorigenic is intended to refer to the teratoma-forming characteristics of undifferentiated human non-differentiated stem cells, such as ES cells, and is not intended to indicate malignancy of any kind. This is because the ES cells do not produce typical malignancies when injected into mice. The removal of the tumor-forming trait simply by direct differentiation and selection is another important step in the advancement of stem cell derivatives from laboratory model to useful human therapy.
    [49] In another embodiment, the invention provides an isolated cell population derived from human pluripotent stem cells having tenninally differentiated cells of the pancreatic developmental lineage. At least 50% of this cell population simultaneously expresses one or fl era of insulin, C-peptide and PDX1.
    [50] In yet another embodiment, the invention provides an isolated non-tumor-forming cell population derived from human pluripotent stem cells.
    [52] Example 1. Cell culture and differentiation
    [53] The general differentiation method is illustrated in a simplistic manner in FIG. 1. N1H-approved hESC lines H1 (WA01) and H9 (WA09) were used between culture rounds 24 to 40. Medium for undifferentiated ESCs consisted of 80% DMEM / F 12 and 20% Knockout serum replacement supplemented with 1 mM L-glutamine, 1% non-essential amino acids, 0.1 mM Z-mercaptoethanol and 4 ng / ml bFGF (all from Invitrogen). hESCs were grown in 6-well plates on a feeder stock of irradiated mouse embryo bridge blasts (MEFs) in either ESC media (control group), ESC media plus 50 ng / ml BMP4 (BMP4 group; R&D Systems), or ESC media plus 50 ng / ml BMP4 plus 300 ng / ml noggin (noggin group; R&D Systems) for 4 days.
    [54] In experiments with MatrigelTM, hESCs were grown on growth factor-poor MatrigelTM (BD Biosciences) instead of MEF and culture media were MEF-conditioned media (CM, control group), CM plus 50 ng / ml BMP4 or CM plus 50 ng / ml BMP4 plus 100 ng / ml bFGF. The colonies were transferred by incubation with 2 mg / ml dispase (Invitrogen), after which the cells were rinsed from the plates, pipetted into small pieces, and filtered through a 70 μm cell filter. The filtrates containing small pieces of colonies were placed in 100 mm untreated suspension culture dish with CM on a shaker for 14 days to form embryoid bodies (EBs). EBs were then seeded in serum-free DMEM / F-12 (17.5 mM glucose) medium with ITS supplement (BD, 5 μg / ml insulin + 5 μg / ml transferrin + 5 ng / ml selenic acid), 10 μM nicotinamide (Sigma), 10 ng / ml FGF7 (R&D), 10 nM exendin-4 (Sigma), and 2 g / L BSA (Sigma) for 14, 21 or 28 days, and then harvested. One fraction of each culture was used for RT-PCR and Q-PCR and the remaining cells were either embedded in OCT (EB 14; Tissue-Tek) or excised on coverslips (EB 14 + 14, EB14 + 21 and EB14 + 28) for immunostaining.
    [55] Example 2. Quantitative PCR and RT-PCR.
    [56] Cellular total RNA was extracted with TriZol (Invitrogen). cDNA was synthesized from 1 μg of total RNA using the Superscript First-Strand Synthesis kit (Invitrogen). Quantitative real-time RT-PCR (Q-PCR) was performed using Assays - 534 150 18 on-demand reagents (Applied Biosystems) on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems) for the following transcripts: foxa2, sox17, brachyury, ngn3, PDX 1, insulin, glucagon, glut2, and an endogenous control, ß-actin. Q-PCR was performed according to the device manufacturer's instructions. Relative quantification was performed using a comparative cycle threshold (C fl procedure recommended by the manufacturer. Times of change were calculated as: ZMCT. Mean AACT values from Q-PCR assays were compared using non-paired, two-tailed Student's t test. P-values of <0.05 was considered significant.
    [57] For non-quantitative RT-PCR, oligonucleotide primer pairs were created against human transcripts using gene bank sequences (see FIG. 12). Primers were selected from two different exons and extended over at least one intron sequence. PCR was performed using HotStarTaq DNA polymerase (Qiagen) and the reaction conditions were as follows: initial denaturation at 95 ° C for 15 min, then cycles of 94 ° C for 30 s, 30 s at hybridization temperature, 1 min at 72 ° C , and a final 10 min at 72 ° C. The primers were hybridized at 53 ° C except for pdx1 (56 ° C), sox17 (55 ° C) and foxa2 (50 ° C; with Qiagen's Q solution). A reverse transcriptase (-RT) control sample was amplified with GAPDH primers in each case, and human adult pancreatic RNA was used as a positive control.
    [58] Example 3. Immunorescence inheritance.
    [59] Immunoassay staining of coverslips was performed as described previously (Kahan, BW et al., Pancreatic precursors and differentiated islet cell types from murine embryonic stem cells: an in vitro model to study islet differentiation. Diabetes 52, 2016-2024 (2003)) . The following primary antibodies were used in the indicated dilutions: PDX1 rabbit antimus serum 124000 (gift from C. Wright); insulin mouse monoclonal 10 μg / ml (ATCC No: HB 124); glucagon mouse monoclonal l: 2000 (Sigma); somatostatin mouse monoclonal 1: 2000 (Novonordisk); Ki-67 mouse monoclonal 1:25 (BD Pharmingen); C-peptide rat monoclonal 123000 (BCBC 1921); Brachyury get antihuman 1:20 (R&D); OCT4 get antimus l: 100 (Santa Cruz); Soxl7 get antihuman 534 150 19 1:40 (R&D); FOXA2 rabbit anti-rat 1: 4000 (gift from R. Costa).
    [60] Example 4. Mesendoderm induction by treatment of hESCs with BMP4 on MEFs
    [61] Process of hESCs differentiation was initiated by treating hESCs with BMP4 on MEFs, as generally illustrated in F IG. It is worth noting that a previous study showed that treatment of hESCs on MatrigelTM with 100 ng / ml BMP4 for 7 days causes almost 100% of the cells to differentiate into human chorionic gonadotropin (hCG) -expressing trophoblast cells (Xu, RH et al., BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nature Biotechnology. 20, 1261-1264 (2002)).
    [62] These results were also reproduced as illustrated in FIG. 2, after 7 days of treatment with BMP4 only 100 ng / ml on Matrigel were cell types that highly expressed hCG and secreted hCG protein (FIGS. 2a and b). In more detail, undifferentiated hESCs grown on Matrigel (MG) were divided into 7 groups and treated with: (1) 10 ng / ml BMP4 (B10); (2) 50 ng / ml BMP4 (B50); (3) 100 ng / ml BMP4 (B100); (4) 10 ng / ml BMP4 + 100 ng / ml bFGF (B10F); (5) 50 ng / ml BMP4 + 100 ng / ml bFGF (B50F); (6) 100 ng / ml BMP4 + 100 ng / ml bF GF (B100F); (7) control (ctrl): without any added growth factors -in 1 day, 4 days and 7 days.
    [64] Figure 2 shows that in contrast to cells treated with BMP4 alone, cells treated with low or intermediate doses of BMP4 (10 ng / ml and 50 ng / ml) for 4 days did not produce hCG or produce hCG protein; instead, the cells expressed significantly higher amounts of brachyury and sox17 transcripts while expressing significantly lower levels of Oct4 transcripts compared to control cells at the same time point (FIG. 2 c-e).
    [65] The cells in cultures not treated with BMP4 were OCT4 + but did not express Brachuyry (FIG: 30-q). These OCT4 staining data are consistent with recently published reports showing that decreased OCT4 expression induces loss of pluripotency and dedifferentiation to trofectoderm while increased OCT4 levels induce differentiation into primitive endoderm and mesoderm (N iwa, H. et ah, Quantitative expression of Oct -3/4 de fi nes differentiation, dedifferentiation or self- renewal of ES cells. Nat. Gener. 24, 372-376 (2000)). The levels of Brachyury and 534 150 21 FoxaZ transcripts support staining data. Both non-quantitative (FIG. 3r) and quantitative RT-PCR (Figs. 3 s and t) show an increase in brachyury and Foxa2 transcripts when cultures on MEFs are treated with Blv fl> 4. The participants saw an approximately 170-fold increase in brachyury gene expression (FIG. 3s) and an approximately 5-fold increase in foxa2 expression with BMP4 treatment (FIG. 3h). Together, these data predict that sequential series of embryonic stages arise from hESCs. First, cells arise that are brachyury + or co-express brachyury and Oct4 (which possibly corresponds to a transition phase), which corresponds to mesendoderm. The cells then undergo a further rapid transition within a few days to co-express FoxA2 and Sox17, while losing brachyury expression.
    [66] Example 5. Several additional necessary components for early cell differentiation against pancreatic developmental lineage from hESCs in vitro are identified.
    [67] To further define culture conditions and media additives that promote the development of pancreatic cells from hESCs in vitro, the researchers evaluated the need for MEFs during the BMP4 treatment period. In cells treated with BMP4 grown on MatrigelTM, transcripts were not detected for any of the genes studied, including brachyuzy, foxa2, pdx] and glut2 (Fig. 4c), whereas they were readily detected in BMP4 treated cells grown on MEFs. In addition, hEBs derived from cells grown on MEFs compared to MatrigelTM were morphologically different (FIG: 4d-g). Cells grown on MatrigelTM resulted in EBs that were smaller, more compact, and more opaque, EBs derived from BMP4-treated cells grown in the presence of MEFs. Thus, MEFs have an important role in pancreatic developmental line determination in this protocol.
    [68] Since a high concentration of bFGF has recently been reported to have an MEF-like effect on the growth of hESCs (Ludwig, TE, et al., Derivation of human embryonic stem cells in de fi ned conditions. Nat. Biotechnol. 24, 185-187 ( 2006) and Levenstein, M_E., Et al, Basic FGF Support of Human Embryonic Stem Cell Self-Renewal (Stem Cells (2005)), investigated whether bFGF could replace MEFs in the culture protocol. To determine this, 50 ng / ml BMP4 and 100 ng / ml 534 150 22 bFGF were added to hESCs grown on MatrigelTM for 4 days, then the cells were harvested for in gene expression assay. Compared with untreated hESCs, brachyury gene expression was 1000-fold greater in BMP4- and bFGF -treated hESCs. EBs derived from BlVlP4 and bFGF-treated cells showed similar inorphologies and levels of brachyury, foxaZ, pdxI and glut2 gene expression comparable to EBs from hESCs grown on MEFs and treated with BMP4 (FIGS. 4c and 4e, 4g), suggesting bFGF plays an early, essential role in endothelial specification and / or cell differentiation towards the pancreatic developmental line.
    [69] To determine whether the inductive effect of BMP4 on mesendoderin formation from hESCs was due to canonical BMP signaling, the researchers tested whether the addition of soluble noggin to the culture could counteract the effect of BMP4 treatment. Gene expression was examined in EB14 by RT-PCR (Fig. 4a) and Q-PCR (Fig. 4b) after treatment of hESCs for 4 days with either only 50 ng / ml BMP4 or BMP4 plus 300 ng / ml noggin. The simultaneous addition of noggin and BMP4 can completely block gene expression of brachyury, sox] 7, foxa2, pdx] and glut2 in BMP4-treated hESC cultures. The effect of noggin was dose dependent (data not shown). It is noteworthy that the transcript levels of foxa2 and glut2 in the noggin-treated cells were even lower than in non-BMP4-treated cells, suggesting that there is a minimal BMP4-like effect present in the hESC medium or generated by the hESCs themselves.
    [70] Example 6. Embryoid body (EB) suspension culture of BMP4-treated hESCs promotes cell differentiation against endodermal and pancreatic developmental pathways.
    [7] Based on the defined role of BMP4 in pancreatic speciation in chicken embryos, the aimers were to determine whether BMP4 treatment would promote differentiation of rhododendral and pancreatic cells from hESCs. EBs were therefore formed from either untreated hESCs or hESCs treated with 50 ng / ml BMP4 for 4 days. The researchers' previous experiments showed that differentiation through an EB stage, in which inductive tissue interdiones can occur in three dimensions among the early embryonic germ leaves, positively affects the development of cells of the pancreatic developmental line with differentiation under two-dimensional conditions (Xu et al, Endoderm and pancreatic lineage differentiation from human embryonic stem cells, Cloning and Stem Cells, 8, 96-107, (2006)).
    [72] Transcript analysis showed no detectable pdx] expression in untreated hESCs.
    [74] In addition, Figures 5 (d-o) show immunostaining of EB14s, where bFGF was added during the EB stage and more than 50% of the cells are stained with PDX1.
    [75] Immunostaining of EB14 cells also showed that hESCs could differentiate into mitotically active pancreatic progenitor cells expressing PDX1 and Ki67 (FIG: Sj-1). Many PDX1 + cells were seen in the BMW-treated group, most of which were also Ki67 +, indicating that these cells were actively proliferating. In addition, a significant proportion of the cells expressing PDX1 also co-expressed Nkx6.1 (FIG.
    [76] The above-mentioned constellation of markers suggests that the cells represent a pancreatic progenitor stage similar to the proliferating pancreatic epithelium that grows and expands in the middle of the fetal period before or around the time of the secondary transition when the betest beta cells are formed. In addition, F numerous F oxA2 + cells were seen in channel-like structures, and some cells were also Ki67 +. These structures formed during the EB stage may play an important role in the induction of PDX1 + cells. In contrast, PDX1 + and FoxA2 + cells were not seen at these stages of hESC differentiation without BMP4 treatment (data not shown).
    [77] Serum is known to have an inhibitory effect on differentiation of the genital endodene from ESCs (D'Amour, KA, et al. (2005)) and development of pancreatic developmental line cells (Gao, R., et al., Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture.
    [78] Example 7. Human ESCs yield cells with PDX1 / lNSULlN / C-PEPTID staining.
    [79] To determine if BMP4-treated hESCs have the ability to become fully differentiated, hormone-containing pancreatic islet cells seeded the 14-day EBs on gelatin-coated coverslips in serum-free ITSFNE medium for culture for an additional 14, 21, and 28 days. Transcriptional profiles over this time course were determined by RT-PCR (FIG. 6) and Q-PCR. After inoculation (EB14 + 14, EB144 + 21 and EB14 + 28), endodern and pancreatic-associated gene transcripts, including foxaZ, pdxI, ngn3, insulin, glucagon and glut2 were highly enriched in BMP4-treated cells compared to untreated cells. . Q-PCR data showed the increasing transcript levels of jbxa2 (18-, 21- and 37-fold increase over time course EB 14 + l4, EBl4 + 21 and EB l4 + 28, respectively, jäintört with untreated EB 14), pdx] (17 9 -, 155- and 54l-fold increase) and glut2 (47-, 97- and 224-fold increase). Essentially, insulin and glucagon transcripts were not detectable in untreated EB14 cells. In contrast, insulin and glucagon mRNA levels in BMP4-treated cultures increased dramatically over time. Q-PCR reaction cycles for insulin mRNA change from 50 (undetectable) iEB14 untreated cells to 28, 27 and 25 cycles in BMP4-treated cells over the time course of post-EB differentiation. For glucagon, the change was from 46 cycles to 22, 21 and 20 cycles, in all trials, β-actin cycle times were about 15 cycles. In contrast to the increasing amount of cellular hormone transcripts, brachyury mRNA decreased continuously over time during these late stages and was not significantly different from iron deficiency with the level in EB 14 untreated cells. Consistent with previous studies with hESCs, the trophoblast marker hCG was expressed in BMP4-treated cells at EB 14 (Xu, R.H., et al. (2002)), but not in EBs generated from hESCs that had not been treated with BMP4. After EBs were seeded in serum-free medium, hCG mRNA levels increased dramatically (FIG. 6).
    [81] Also consistent with previous observations in embryos and mESCs, the neuroectoderm macrophage sax] was not detected in BMP4-treated cells, whereas it was induced in differentiation cultures initiated with untreated hESC-534 150 26 colonies (FIG. 6). These data suggest that: 1) serum-free conditions promote cellular horn gene expression at the expense of trophoblast differentiation (eg, retained cellular gene expression over time compared to lost hCG gene expression over time in serum-free conditions); 2) neural differentiation is likely to be inhibited by BMP4 treatment and / or neural precursors are not selected by BMP4 treatment, 3) serum-free conditions prevent survival and / or expansion of Oct4-expressing undifferentiated cells, and 4) TAT-expressing cells, such as liver cells are supported or is not maintained by these serum-free conditions.
    [82] Consistent with gene expression results, immunostaining data show that BMP4-treated cultures contained large clusters of PDX1 + cells after seeding. In contrast to PDX1 + found in EBs, most cells no longer stained with Ki67 (FIG. 7d-í), suggesting that the cells had begun terminal differentiation. The cells that stained for either PDX1 or insulin, or PDX1, insulin and C-peptide emerged within the large clusters of PDX1 + cells. At 14 after EB seeding, 5 of 17 (29%) EBs showed insulin + cells that increased over time so that at 28 days after seeding, 16 (63%) of EBs contained insulin + cells and the average number of positive cells per EB increased. The fact that the recipients observed numerous insulin / C-peptide immunostained cells, which always co-express PDX1 and are located in the PDX1 + clusters, as well as a significant increase in insulin mRNA suggest a phenotypic pattern of true ß cells. Glucagon + and somatostatin + cells did not co-target for C-peptide as would be expected of adult endocrine cell types.
    [83] Example 8. EpCAM-based MACS sorting further enriched cultures for endoderm and pancreatic cells.
    [84] The inventors discovered that when BMP4 / bFGF-treated cultures are stained for EpCAM and PDX1, numerous cell clusters containing cells that co-express both markers can be seen (FIG. 10a). This detection provided an opportunity to preferentially select PDX1 + cells based on EpCAM expression and MACS sorting. To test this hypothesis, cells differentiated according to the BMP4 / bFGF treatment protocol are labeled with an anti-EpCAM antibody labeled with an appropriate secondary antibody and sorted. Following EpCAM + sorting of differentiated hESC progeny, the transcript levels of sox] 7 and foxa2 increased, as did the levels of pdxI and EpCAM (FIG. 10b). In this regard, MACS sorting for EpCAM can be used to enrich or purify differentiated ESC cultures to eliminate non-endodermal cells arising from non-selective culture conditions. Sorting for EpCAM results in a population in which ~ 95% of the cells are EpCAM + (FIG. 7c). The inventors hypothesize that by combining two or two rounds of sorting (selection for EpCAM and removal of SSEA3 or 4+ cells), the cultures of endoderm / pancreatic progenitor cells will be further enriched while removing undifferentiated, teratoma-forming cells from the cultures.
    [86] The isolated populations of endodermal or pancreatic progenitor populations may improve therapy for adults. For example, these progenitor cells can be co-transplanted with human adult islets or hepatocytes. These progenitor populations can also be transplanted separately, without first going through the terminal differentiation step. In this therapeutic strategy, it is expected that the progenitor cells will differentiate in the patient into the desired functional cells or tissues. Whole tissue regeneration can also be imagined from such endodermal or pancreatic progenitor cells.
    [87] Description of each patent, patent application and publication cited herein is hereby incorporated by reference in its entirety.
    [88] It is to be understood that certain adaptations of the invention described in this specification are a matter of routine optimization to those skilled in the art, and may be implemented without departing from the spirit of the invention, or the scope of the appended claims.
    权利要求:
    Claims (23)
    [1]
    A method of culturing human pluripotent stem cells to produce cells of the pancreatic developmental line comprising the steps of: (a) culturing the stem cells in conditions that induce differentiation in the mesendodernal direction, wherein the conditions include the presence of an effective amount of bone morphogenesis protein; (b) culturing the cells of step (a) in conditions conducive to the formation of intact embryoid bodies, wherein said embryoid bodies are surrounded by a layer of visceral yolk sac; and (c) culturing the cells from the embryoid bodies according to step (b) in conditions that favor terminal differentiation of the cells towards the pancreatic developmental line.
    [2]
    The method of claim 1, wherein the culture conditions in step (c) include culturing the embryoid body cells in a serum-free medium containing insulin, transferrin, selenium, FGF7, nicotinamide and exendin-4.
    [3]
    The method of claim 1, wherein the amount of bone morphogenesis protein ranges from about 10 ng / ml to about 100 ng / ml.
    [4]
    The method of claim 1, wherein the bone morphogenesis protein is BMP4.
    [5]
    The method of claim 4, wherein an effective amount of BMP4 is about 50 ng / ml.
    [6]
    The method of claim 1, wherein the stem cells of step (a) are further cultured in the presence of an effective amount of a bl broblast growth factor to induce mesendodennal direction.
    [7]
    The method of claim 6, wherein the amount of β-broblast growth phalas is in the range of from about 10 ng / ml to about 200 ng / ml.
    [8]
    The method of claim 7, wherein the fi bridge blast growth factor is bFGF. 534 150 30
    [9]
    The method of claim 8, wherein the effective amount of bFGF is about 100 ng / ml.
    [10]
    The method of claim 1, further comprising the step of (d) selecting the cells of step (c) that are positive for the expression of the epithelial cell adhesion marker (EpCAM) to retain cells of the pancreatic developmental line that show a decrease in tumorigenicity.
    [11]
    11. ll. The method of claim 10 wherein the selection is performed by magnetically activated cell sorting.
    [12]
    The method of claim 1, wherein the mesendoderm cells co-express Oct4 and Brachyury (T) in individual cells.
    [13]
    The method of claim 1, wherein the embryoid bodies include definitive endoderm cells with channel-like structures containing Foxa2 +, Sox1 7+ and PDX1 + cells.
    [14]
    The method of claim 1, wherein the terminally differentiated cells simultaneously express insulin, C-peptide and PDX1.
    [15]
    A method for sequentially enriching a culture derived from human pluripotent stem cells with respect to cells of endodermal and pancreatic developmental lines, the method comprising the steps of: (a) culturing the stem cells in conditions that induce differentiation in the mesendodernal direction, wherein the conditions include presence of an effective amount of a bone morphogenesis protein and fi broblast growth factor; (b) culturing the cells of step (a) in conditions conducive to the formation of intact embryoid bodies, wherein said embryoid bodies are surrounded by a layer of visceral yolk sac; (C) culturing the cells from the embryoid bodies according to step (b) in conditions that favor terminal differentiation of the cells towards the pancreatic developmental line.
    [16]
    The method of claim 15, wherein the culture conditions of step (c) include culturing the embryoid body cells in a serum-free medium containing insulin, transferrin, selenium, FGF7, nicotinamide and exendin-4.
    [17]
    The method of claim 15, further comprising the step of (d) selecting the cells of step (c) that are positive for the expression of the epithelial cell adhesion marker (EpCAM) to retain cells of the pancreatic developmental line that show a decrease in tumorigenicity.
    [18]
    The method of claim 17 wherein the selection is performed by magnetically activated cell sorting.
    [19]
    The method of claim 15, wherein an effective amount of bone morphogenesis protein ranges from about 10 ng / ml to about 100 ng / ml and an effective amount of fibroblast growth factor is in the range of from about 20 ng / ml to about 200 ng / ml.
    [20]
    A method of culturing human pluripotent stem cells to produce a cell population of the pancreatic developmental line that does not have tumorigenic properties, the method comprising the steps of: (a) culturing the stem cells in conditions that induce differentiation toward mesendoderm, wherein the conditions include presence of an effective amount of a bone morphogenesis protein; (b) culturing the cells of step (a) in conditions that cause the formation of intact embryoid bodies, wherein said embryoid bodies are surrounded by a layer of Visceral yolk sac; (c) culturing the cells from EBs according to step (b) in conditions that favor terminal differentiation of the cells toward the pancreatic developmental line; and 534 150 32 (d) selects for expression a cell surface marker indicating lineage to a particular differentiated developmental line, wherein the marker is EpCAM, wherein the resulting cell culture does not form teratomas upon injection into immunocompromised mice.
    [21]
    The isolated cell population of claim 20, step (b), wherein the embryoid bodies include definitive endodoric cells with channel-like structures containing Foxa2 +, Sox17 + and PDX1 + cells.
    [22]
    The isolated cell population of claim 20, step (c), wherein the differentiated cells co-express PDXI in combination with insulin and C-peptide.
    [23]
    The isolated cell population of claim 20, step (d), wherein the differentiated cells express EpCAM and do not form teratomas upon injection into immunocompromised mice.
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    同族专利:
    公开号 | 公开日
    AU2007248609B2|2012-11-01|
    GB2452186B|2011-01-26|
    EP2027258A2|2009-02-25|
    CA2650561C|2014-02-25|
    GB0821641D0|2008-12-31|
    CA2650561A1|2007-11-15|
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    WO2007130474A2|2007-11-15|
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    US20070259423A1|2007-11-08|
    WO2007130474A3|2008-01-03|
    SE0850111L|2009-01-21|
    US20110081720A1|2011-04-07|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    US79666206P| true| 2006-05-02|2006-05-02|
    PCT/US2007/010662|WO2007130474A2|2006-05-02|2007-05-02|Method of differentiating stem cells into cells of the endoderm and pancreatic lineage|
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